General guidelines for siRNA design are outlined below.

Choice of a target sequence and oligonucleotide design

Initial studies of mammalian RNAi suggested that efficient gene silencing is achieved using double-stranded siRNA having a 19 nt double-stranded complementary region and a 2-nucleotide 3' overhang at each end (see examples on next page). Current models of the RNAi mechanism suggest the antisense siRNA strand recognizes the specific gene target.

1. Choose a sequence in the coding region of the mRNA with a GC ratio as close to 50% as possible (see examples below). Ideally the GC ratio will be between 45% and 55%. An siRNA with 60% GC content has worked in many cases. However, when an siRNA with 70% GC content is used for RNAi, a sharp decrease in the level of silencing is typically observed. Avoid regions within 50–100 nt of the AUG start codon or 50–100 nt of the termination codon. If the desired target region begins with AA, choose an additional 19 nt of the coding sequence of the mRNA to determine the %GC. When the sense siRNA is synthesized, it will be created with a dTdT 3' end (Example 1). If the target region does not begin with AA choose a 23 nt region of the coding region of the gene to determine the %GC (Example 2)
2. Avoid more than three guanosines or three cytosines in a row. Poly G and poly C sequences can hyperstack and therefore form agglomerates that potentially interfere in the siRNA silencing mechanism
3. Where possible, choose target sequences that start with two adenosines (Example 1). This will make synthesis easier, more economical, and create an siRNA that is potentially more resistant to nucleases. When a sequence that starts with AA is used, siRNA with dTdT overhangs can be produced. If it is not possible to find a sequence that starts with AA and satisfies considerations 1 and 2, choose any 23 nt region of the coding sequence with a GC content between 45 and 55% that does not have more than three guanosines in a row (Example 2).
4. Ensure that your target sequence is not homologous to any other genes. It is strongly recommended that a BLAST search of the target sequence be performed to prevent the unwanted silencing of genes that contain an identical sequence.
5. Based on feedback from customers, labeling the 3'-end of the sense strand leads to the least interference in the gene silencing effectiveness of synthetic siRNA.

When these considerations are followed, RNAi effectively silences genes in more than 80% of cases. Current data indicates there are regions of some mRNAs where gene silencing does not work efficiently. To help ensure that a given target gene is silenced, it is recommended that at least two target sequences — as far apart on the gene as possible are chosen. The above considerations do not take into account mRNA secondary structure. At present, it does not appear that mRNA secondary structure has a significant impact on gene silencing. Some examples of the different types of target sequence motifs used and the resulting siRNA are given below.

Example 1: AA(N19) sequence motif

Note: Experiments have shown that the 3'-overhang on the sense strand does not
contribute to specificity. Therefore, an AA(N19) target sequence is sufficient for the design
of a 21mer siRNA duplex.

Example 2: N23 sequence motif (45–50% GC, no poly G)

Note: To create a 21 base pair duplex which has overhangs that match the target
sequence and a 19 base pairing region, a target sequence of 23 bases is needed.

Example 3: AA(N19)TT sequence motif

Note: This approach drastically limits target selection.